EP0138133A1 - Clones du cADN codant pour des polypeptides avec activité du facteur de croissance non-spécifique à ligne cellulaire (multi-CSF) et/ou du facteur de croissance de mastocytes (MCGF) - Google Patents
Clones du cADN codant pour des polypeptides avec activité du facteur de croissance non-spécifique à ligne cellulaire (multi-CSF) et/ou du facteur de croissance de mastocytes (MCGF) Download PDFInfo
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- EP0138133A1 EP0138133A1 EP84111677A EP84111677A EP0138133A1 EP 0138133 A1 EP0138133 A1 EP 0138133A1 EP 84111677 A EP84111677 A EP 84111677A EP 84111677 A EP84111677 A EP 84111677A EP 0138133 A1 EP0138133 A1 EP 0138133A1
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- growth factor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/12—Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/14—Lymphokine; related peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/14—Lymphokine; related peptides
- Y10S930/141—Interleukin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/30—Signal or leader sequence
Definitions
- This invention relates generally to the application of recombinant DNA technology to elucidate the control mechanisms of the mammalian immune response and, more particularly, to the isolation of deoxyribonucleic acid (DNA) clones coding for polypeptides exhibiting multi-lineage cellular growth factor activity and/or mast cell growth factor activity.
- DNA deoxyribonucleic acid
- Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment.
- the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product.
- cDNA complementary DNA
- mRNA messenger RNA
- Tfie carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
- eukaryotic proteins so- produced include:
- Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the basic differentiation of pluripotential hematopoietic stem cells into the vast number of progenitors of the diverse cellular lineages responsible for the immune response.
- Cell lineages important in this response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T cells (of various subsets) that induce or suppress B cells and some of the other cells (including other T cells) making up the immune network.
- mast cell a granule-containing connective tissue cell located proximate to capillaries throughout the body, with especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts.
- Mast cells play a central role in allergy-related disorders, particularly anaphylaxis, and this role can be briefly stated as follows: once certain antigens crosslink special immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases the mediators (e.g., histamine, serotonin, heparin, kinins, etc.) which cause allergic reactions, e.g. anaphylaxis.
- mediators e.g., histamine, serotonin, heparin, kinins, etc.
- the pluripotent stem cell which can repopulate a lethally irradiated animal for most, if not all, immunological cell types (e.g., red cells, platelets, lymphocytes, various granulocytes and monocytes/macrophages).
- the pluripotent cell not only has the capacity to regenerate the pluripotent stem cell compartments (self-renewal), but also gives rise to progenitor cells committed to development along one particular lineage pathway. Progeny of a particular committed stem cell appear to share the same lineage commitment as the parent cell (Metcalf, D., "Hemopoietic Colonies", Springer Publishing Co., New York, N.Y. [1977]).
- CSF colony stimulating factor
- multi-CSF multi-lineage cellular growth factor
- interleukin-1 IL-1
- IL-2 interleukin-2
- IL-3 interleukin-3
- IL-3 an important characteristic of IL-3 is its ability to support the growth of cell lines having the phenotypic characteristics of mast cells (Ihle, J. et al., Immunological Rev. 63: 5-32 [1982]). A number of other cellular growth properties have been ascribed to IL-3 as well (see Ihle, J. et al., J. Immunol. 131: 282-287 and 129: 2431 [1981]), but its precise relationship with multi-CSF has been unclear.
- mouse IL-2 and IL-3 have been at least partially characterized biochemically (Gillis, S. et al., J. Immunol. 124: 1954-1962 [1980] and Ihle, J. et al., J. Immunol. 129: 2431-2436 [1982], respectively)
- IL-2 is presently the accepted primary factor responsible for T-cell growth, whereas the protein(s) responsible for mast cell growth factor (MCGF) and CSF activity have not been agreed upon to the same extent.
- MCGF mast cell growth factor
- mouse IL-2 has a molecular weight (probably as a dimer) of approximately 30-35,000 (Simon, P. et al., J. Immunol.
- the present invention provides cDNA clones coding for polypeptides exhibiting mammalian mast cell growth factor (MCGF) activity and or multi-lineage cellular growth factor activity.
- MCGF mammalian mast cell growth factor
- a nucleotide sequence for a cDNA and a putative amino acid sequence for an associated polypeptide are shown in Figure 1.
- the cDNA sequence can be integrated into various vectors, which in turn can direct the synthesis of the corresponding polypeptides in a variety of hosts, including eukaryotic cells, such as mammalian cells in culture.
- the invention provides a process for producing a polypeptide exhibiting mammalian MCGF activity and/or multi-lineage cellular growth factor activity, the process comprising the steps of:
- the cDNA sequences are derived from an mRNA sequence coding for the polypeptides, and the host is an organism such as a eukaryotic, e.g. mammalian, cell transfected or transformed with the vector.
- the vector preferably comprises also a second nucleotide sequence capable of controlling expression of the nucleotide sequence coding for the polypeptide.
- This second sequence coding can include a promoter sequence, one or more intron sequences and a polyadenylation sequence, to permit, respectively, transcription, splicing and polyadenylation of the nucleotide sequence coding for the polypeptide.
- the vector when the host is a mammalian cell, such as a COS-7 monkey (kidney) cell, the vector contains the promoter sequence of the simian virus 40 (SV40) early region promoter and the polyadenylation sequence of the SV40 late region polyadenylation sequence.
- SV40 simian virus 40
- the mouse cDNA sequence of Figure 1 (see below) is capable of hybridizing with other DNA sequences, such as DNA coding for other mammalian growth factors from a cDNA or genomic library. It is noted that the described cDNA sequences seem to contain information for a leader sequence.
- polypeptides of the present invention are capable of enhancing mammalian mast cell and other cell growth, particularly in in vitro cultures.
- Suitable pharmaceutical compositions can be prepared by adding the polypeptides (preparations of which are essentially free of other mammalian growth factors) to therapeutically compatible carriers.
- transcription of the 950 bp cDNA insert contained in the pcD expression vector from the SV40 early promoter is indicated by the arrow.
- the locations of the splice donor and acceptor sites are shown.
- the polyadenylation signal, also derived from SV40, is located at the 3'-end of the cDNA insert.
- the cDNA insert is heavily shaded.
- the remainder of the vector sequences are derived from pBR322, including the S-lactamase gene (Amp R ) and the origin of replication.
- complementary DNA (cDNA) clones are provided for polypeptides exhibiting mammalian mast cell growth factor (MCGF) activity and/or multi-lineage cellular growth factor (multi-CSF) activity.
- MCGF mammalian mast cell growth factor
- multi-CSF multi-lineage cellular growth factor
- FIG. 1 An exemplary, putative amino acid sequence based on the isolated nucleotide sequence is shown in Figure 1.
- a portion of the predicted sequence (amino acids 33 to 41) is identical with the reported NH 2 - terminal sequence of mouse Interleukin-3 (IL-3), which has been shown to exhibit mouse MCGF activity and multi-CSF activity (Ihle, J. et al., J. Immunol. 129, 2431-2436 [1982]; Ihle, J. et al., J. Immunol. 131, 282-287 (1983); and Garland, J. et al., Eds. Oppenheim, J. and Cohen, S., "Interleukins, Lymphokines, and Cytosines", Proceedings of Third Int.
- IL-3 mouse Interleukin-3
- the coding region located between the translation start codon (ATG) and the beginning of the sequence contained in IL-3 is rich in hydrophobic amino acids, as would be expected for a leader sequence of a secreted protein. Therefore, the polypeptide's mature form in vivo, as secreted, possibly begins with an Asp residue, as does IL-3, and the preceding 20 or so amino acids - constituting the putative leader sequence - are removed by proteolytic processing. Assuming this to be accurate, the mature polypeptide exhibiting MCGF and multi-CSF activities would consist of 134 amino acids, with a calculated molecular weight of about 15,000.
- one of the cDNA clones of this invention directs the synthesis of biologically active MCGF and multi-CSF.
- Addition of this expressed cloned gene product to cultures of mouse bone-marrow cells allows the expansion of hematopoietic cells committed to multiple lineages; it supports the formation of burst-forming erythroid colonies (BFU-E), granulocyte/macrophage colonies (CFU-G/M), mast cell colonies (CFU-mast), as well as colonies of multiple lineages (CFU-Mixed), and sustains multipotential stem cells (CFU-S) in liquid culture.
- cDNAs of the present invention A variety of methods may be used to prepare the cDNAs of the present invention.
- total mRNA is extracted (e.g., as reported by Berger, S. et al., Biochemistry 18: 5143-5149 [1979]) from a cell line (e.g. a hybrid cell line) producing polypeptides exhibiting mammalian mast cell growth factor activity.
- the double-stranded cDNAs from this total mRNA can be constructed by using primer-initiated reverse transcription (Verma, I., Biochim. Biophys. Acta, 473: 1-38 [1977]) to make first the complement of each mRNA sequence, and then by priming for second strand synthesis (Land, H.
- the cDNAs can be cloned by joining them to suitable plasmid or bacteriophage vectors (Rougeon, F. et al., Nucleic Acids Res., 2, 2365-2378 [1975] or Scherer, G. et al., Dev. Biol. 86, 438-447 [1981]) through complementary homopolymeric tails (Efstratiadis, A. et al., Cell, 10, 571-585 [1977]) or cohesive ends created with linker segments containing appropriate restriction sites (Seeburg, P.
- a preferred method of obtaining the full-length cloned cDNAs of this invention is the procedure developed by H. Okayama and P. Berg (Mol. and Cell. Biol., 2: 161-170 [1982]).
- This method has the advantage of placing the cDNA inserts in a bacterial cloning vector at a position whereby the cDNA can also be directly translated and processed in mammalian cells. Briefly, the first cDNA strand is primed by polydeoxythymidylic acid covalently joined to one end of a linear plasmid vector DNA. The plasmid vector is then cyclized with a linker DNA segment that bridges one end of the plasmid to the 5' end of the cDNA coding sequence.
- the cDNA can be expressed in vitro in COS-7 mouse kidney cells without further modification.
- SV40 Simian Virus 40
- linker containing a modified SV40 late region intron See generally Okayama, H. and Berg, P., Mol. and Cell. Biol., 3: 280-289 [1983] and Jolly, D. et al., Proc. Nat. Acad. Sci. U.S.A., 80: 477-481 [1983].
- the desired cDNA clones can also be detected and isolated by hybridization screening with appropriate mRNA samples (Heindell, H. et al., Cell, 15: 43-54 [1978]).
- the cDNA libraries can be screened by hybrid selection (Harpold, M. et al., Nucleic Acid Res., 5: 2039-2053 [1978] or Parnes, J. et al., Proc. Nat. Acad. Sci. U.S.A., 78: 2253-2257 [1981]) or in Xenopus oocytes (Aurdon, J., Nature, 233: 177-182 [1971]).
- the cDNA clones are collected, and random pools are checked for the presence of the desired cDNAs by hybrid selection, translation, and assay (e.g. by measuring MCGF or multi-CSF growth factor activity, the existence of antigenic determinants, or other biological activities). Pools positive by these criteria can then be probed with an appropriate subtracted probe, e.g., cDNA from a B cell line and/or uninduced T cell line. Thereafter, the positive, probed pools are divided into individual clones which are tested by transfection into a suitable host (such as a mammalian cell culture), and the host supernatant assayed for the desired activity (e.g. multi-CSF or MCGF activity). Positive clones are then sequenced.
- a suitable host such as a mammalian cell culture
- the mast cell and other lines will be considered first, followed by general descriptions of the procedures of the in vitro translation of mRNA coding for a protein exhibiting MCGF activity; the construction of a cDNA library containing the cDNA sequences; hybrid selection of the library; isolation of full-length cDNA clones in a plasmid vector and subsequent expression in mammalian cells; multi-CSF assays; human multi-CSF and MCGF isolation, subcloning and expression in bacteria and yeast; and purification and formulation. A more detailed description of the entire experimental process will follow thereafter.
- the preferred cells for use in connection with the present invention are those developed as described by Galli, J. et al. (J. Cell Biol., 95: 435-444
- One cloned mast cell line designated MC/9 and deposited at the American Type Culture Collection (accession number ATCC CRL 8306) was grown in Dulbecco's modified Eagle's medium (DME) supplemented with 5% supernatants from a ConA-activated T-cell line, designated Cl.Ly 1 + 2 - /9 and deposited at the American Type Culture Collection (Accession Number ATCC CRL 8179) (Nabel, G. et al., Nature, 291: 332-334 [1981]).
- DME Dulbecco's modified Eagle's medium
- This T-cell line was derived from C57Bl/6 mice (Nabel, G. et al., Proc. Natl. Acad. Sci.
- the MC/9 cells are used to assay for MCGF activity, preferably by a 3 H-thymidine incorporation assay according to established methods (e.g., Nabel et al., Nature, 291: 332-334 [1981]). Briefly, MC/9 cells (10 4 /well) are cultured in flat bottom Falcon microtiter trays in DME supplemented with 4% fetal calf serum, 50 PM 2-mercaptoethanol (2-ME), 2mM glutamine, non-essential amino acids, essential vitamins and varied concentrations of supernatant in a final volume of 0.1 ml. To each culture is added 0.5 Ci 3 H-thymidine for the last 4 hr of a 24 hr incubation period. The cells are then harvested onto glass filters and the radioactivity measured by liquid scintillation spectrometer.
- 3 H-thymidine incorporation assay e.g., Nabel et al., Nature, 291: 332-334 [1981
- Total cellular mRNA can be isolated by a variety of well-known methods, e.g., by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (Biochemistry, 18: 5294-5299 [1979]). If this method is used, approximately 100 ug of polyA + mRNA, selected on columns of oligo (dT) cellulose, is obtained from 1-2 x 10 8 activated helper T-cells, such as Cl.Ly 1 + 2 - /9.
- RNA is precipitated with 2 volumes of ethanol.
- Filter hybridizations are preferably performed essentially as described by Parnes et al. (Proc. Natl. Acad. Sci. U.S.A., 78: 2253-2257 [1981]). Aliquots of eluted mRNA are injected into individual Xenopus laevis oocytes by methods well known in the art. Supernatants from viable oocytes are collected after 48 hr, pooled and assayed for activities.
- the cDNA library can best be constructed using the pcDV1 vector-primer and the pL1 linker fragment [available from P-L Biochemicals Inc., Milwaukee, WI) according to procedures which result in greatly enriched full-length copies of mRNA transcripts (e.g. Okayama, H. and Berg, P., Mol. Cell Biol., 2, 161-170 [1982] and Mol. Cell Biol., 3, 280-289 [1983]).
- the plasmid vector which contains SV40 early promoter and SV40 RNA processing signals, is designed to promote expression of the cloned cDNA segment in mammalian cells.
- the cyclized vector-cDNA preparation is transformed into a competent bacterial cell, such as E. coli MC1061 cells (Casadaban, M. and Cohen, S., J. Mol. Biol., 138: 179-207 [1980]) using calcium chloride (Cohen, S. et al., Proc. Nat. Acad. Sci. U.S. A ., 69: 2110-2114 [1972]).
- E. coli MC1061 cells Casadaban, M. and Cohen, S., J. Mol. Biol., 138: 179-207 [1980]
- calcium chloride Cohen, S. et al., Proc. Nat. Acad. Sci. U.S. A ., 69: 2110-2114 [1972].
- 5 Pg of polyA + RNA from ConA-stimulated Cl.Ly 1 + 2 - /9 cells about 1.5 x 10 6 independent transformants are obtained.
- clones are picked up individually and inoculated into wells of microtiter plates (Flow Laboratories Inc., McLean, Virginia) containing 200 ⁇ l of L-broth, 50 ⁇ g/ml of ampicillin, and 7% DMSO. If desired, sublibraries based on the size of cDNA insert are prepared from total cDNA library as described by Okayama, H. and Berg, P. (Mol. Cell Biol., 3, 280-289 [1983]). Briefly, plasmid DNA is digested with SalI, ClaI, and HindIII separately, and electrophoresed in 1% agarose gel.
- the gel is sliced into 7 sections corresponding to cDNA insert sizes of 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, and more than 6 kilobases (kb). DNA is extracted from each slice, recyclized with T4 DNA ligase, and used to transform MC1061. All nucleotide sequencing can be performed according to the procedure of Maxam, A. and Gilbert, W. (Methods Enzymol., 65: 499-560 [1980]).
- a 32 P -cDNA probe is enriched for ConA-induced sequence by two cycles of cDNA absorption in order to remove cDNA sequences common between Cl.Ly 1 + 2 - /9 and closely related, but differentiated, cells of the immune system, such as B cell myelomas (see Davis, M. et al., "Isolation of B4 T-Cell Specific Genes", Vitteta, E. and Fox, C. eds., UCLA Symp., pg. 48 [1982]).
- mRNA having MCGF or multi-CSF activity from a sucrose gradient fraction is preferably used as template for reverse transcriptase using oligo (dT) 12-18 primers (available from Collaborative Research, Waltham, Mass.).
- oligo (dT) 12-18 primers available from Collaborative Research, Waltham, Mass.
- the single-stranded 32 P-cDNA enriched for ConA-induced sequences, constituting approximately 1-2% of the starting material, is then used for colony hybridization (Maniatis, T. et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, U.S.A. [1982]).
- Multi-CSF activity comprises testing for the ability to act on multipotential progenitor cells, or a number of lineage restricted cells, or both.
- the assay conditions allow generation of burst-forming erythroid colonies (BFU-E), granulocyte/macrophage colonies (CFU-G/M) and colonies of mixed lineages (CFU-Mixed) and are performed generally according to the procedures of Metcalf, D. et al. (J. Cell Physiol., 98: 401-420 [1979]) and Johnson, G. (J. Cell Physiol., 103: 371-383 [1980]).
- Bone marrow cells can be harvested from the femurs of C57B1/6 mice. The cells are washed once and a single-cell suspension prepared in Iscove's modified Dulbecco's Medium, [IMDM] (GIBCO, Grand Island, New York) + 3% Fetal Calf Serum [FCS] (GIBCO). The single-cell suspension is plated in plastic tissue-culture dishes and incubated 1-2 hours in a 37°C incubator with 6% C0 2 to allow cells to adhere to the dish. The non-adherent cells are then removed and in some cases placed over a discontinuous Percoll (Sigma Chemical Co., St.
- CFU-c's can be assayed by using a modification of the methyl-cellulose procedure of Iscove et al. (J. Cell Physiol., 83: 309 [1974]).
- FCS final concentration 25%
- 2-mercaptoethanol 5 x 10 -5 M
- penicillin-streptomycin (1:100 of GIBCO stocks)
- methyl-cellulose (1.1%, 4000 centipoise
- cells (1.5-2 x 10 5 /ml) and various experimental factors to be tested for CFU-c ability (30%)
- the plates are incubated 7 days in a 37°C/6% C0 2 incubator. They are then scored for colonies using a dissecting microscope (4x).
- a colony is defined as consisting of 50 or more cells. Individual colonies can be extracted, placed on microscope slides, fixed and stained with Wright/Geims (See Todd-Sanford, Clinical Diagnosis By Laboratory Methods, 15th edition, Davidsohn and Henry (eds.) 137 [1974]). Morphological analysis of cell types present per single colony is then determined.
- BFU-E Burst Forming Unit - Erythroid or CFU-E
- Bone marrow is extracted from femur bones of C57B1/6 mice.
- Cells are washed twice with Dulbecco's modified Eagle's medium [ D M E] (GIBCO) and either injected immediately into the tail vein of lethally irradiated (1000 rads) C57B1/6 recipients or treated further.
- Treatment consists of the following various procedures: 1) lysis of the cells with anti-0 and complement, followed either by immediate injection into recipients, or by culture for various times under various conditions before injection; 2) alternatively, no antiserum lysis is performed, and cells are placed immediately into culture under various conditions.
- the culture conditions can be as follows: cells are resuspended at 1 x 10 6 cells/ml in medium consisting of DME (GIBCO) + 2-ME (5 x 10 -5 M), MEM-Vitamins (1:100) (GIBCO), non-essential amino acids (1:100) (GIBCO), L-glutamine (1:100) (GIBCO), penicillin/streptomycin (1:100) (GIBCO), a mix of arginine, asparagine, and folic acid, 15% FCS (GIBCO), 2mM sodium pyruvate + various factors to be tested for maintenance of CFU-s (final concentration 25%).
- This cell preparation is then plated in 24 well tissue culture plates (Falcon) at 1 ml/well and incubated in a 37°C/10% C0 2 incubator for various times (minimum of 7 days). Every 3-4 days, non-adherent cells are removed, spun down, resuspended in fresh media containing the appropriate factor, and replated. For assays in which incubation lasted more than 7 days, cells are "moved up" to larger plastic tissue culture vessels in order to maintain all non-adherent cells at a concentration not exceeding 5 x 10 5 per ml.
- DNA clones of rodent genes have been used to identify and isolate DNA encoding the homologous human genes. Because of the relatively low degree of homology between human and rodent genes, the stringency of hybridization conditions must be adjusted to allow for cross-hybridization between sequences which are only 75-80% homologous.
- Several different experimental protocols have been used to achieve this purpose. For example, the human CK immunoglobulin light chain gene has been isolated using the corresponding mouse CK gene as a probe (Heiter, P. et al., Cell 22: 197-207 [1981]) and mouse transplantation antigen genes have been isolated by hybridization to DNA clones encoding their human counterparts (Steinnetz, T. et al., Cell 24: 125-134 [1981]).
- a preferred method entails plating Y phage clones from a library of human genomic DNA (Maniatis, T. et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, U.S.A. [1982]) at a density of 2 x 10 4 to 5 x 10 4 plaques per 150 mm plate on an appropriate host strain, such as E, coli LE392. Ten to twenty plates are generally sufficient.
- the plates are refrigerated for two hours and then a 132 mm nitrocellulose filter is applied to the agar surface of each plate.
- the filter is allowed to remain in contact with the plate for at least five minutes, during which time the filters are keyed to the plates by puncturing with an ink-filled 22-gauge needle.
- the filters are then peeled from the plates and incubated successively for at least two minutes first in 250 ml of 0.1 N NaOH, 0.5 M NaCl; then in 250 ml of 0.5 M Tris ⁇ HCl pH 7.5, 1.5 M NaCl.
- the filters are dried on paper towels and then baked at 80°C for 4-8 hours.
- the filters are wetted in 1x SET (0.15 M NaCl, 30 mM Tris ⁇ HCl pH 8.0, 1 mM Na 2 EDTA), then incubated in a solution of 3x SET, 5x Denhardt's (Denhardt, D.T., B.B.R.C. 23: 641-646 [1966]), 10% dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), and 50 ⁇ g/ml each poly (rA), poly (rC), and poly (rG), at 65°C for 2 hrs (1.5-2 ml/filter) with constant agitation.
- 1x SET 0.15 M NaCl, 30 mM Tris ⁇ HCl pH 8.0, 1 mM Na 2 EDTA
- Hybridizing plaques are picked from the agar plates with sterile pasteur pipets, and each is expelled into 1 ml of 0.1 M NaCl, 0.01 M Tris ⁇ HCl pH 7.5, 10 mm MgCl 2 , 100 ⁇ g/ml gelatin, with 50 P l of CHC1 3 added. After at least 4-8 hours in the cold, the phages from each plaque are rescreened at low density (2000-4000 plaques/150 mm plate) by a procedure identical to that described above.
- human cDNA library should be prepared using RNA from an appropriate cellular source, such as human peripheral blood T lymphocytes (see Gray, P. et al., Nature 295: 503-508 [1972]).
- Full length cDNA clones can be identified by expression in Cos-7 cells, again as was done for the mouse cDNA clones.
- the isolated human multi-CSF cDNA clones will be able to express a factor capable of stimulating human bone marrow cells.
- Prokaryotes such as E. coli are very suitable for expression of the polypeptides of the present invention (see, for example, U.S. patents number 4,338,397 and 4,411,994), provided that glycosylation is not desired.
- promoters should be utilized, such as the S-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature, 275: 615 [1978]; Itakura et al., Science, 198: 1056 [1977]; Goeddel et al., Nature 281: 544 [1979] or a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8: 4057 [1980]). These are the most commonly used promoters, but other microbial promoters are available.
- yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7: 149 [1968]; Holland et al., Biochemistry, 17: 4900 [1978]).
- Other promoters that have the additional advantage of transcription controlled by growth conditions may be used.
- any plasmid vector containing a yeast-compatible promoter, an origin of replication and termination sequences is suitable.
- cell cultures derived from multicellular organisms may also be used as hosts.
- useful host cell lines are HeLa cells, Chinese hamster ovary cell lines, and baby hamster kidney cell lines.
- Expression vectors for such cells ordinarily include, as necessary, an origin of replication, a promoter located in front of the gene to be expressed, along with any required ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
- the expression vector often has control functions provided by viral material.
- commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently SV-40. (See generally U.S.P. 4,399,216, WO 81/02425 and WO 83/03259.)
- cDNA clones of the present invention in E. coli, suitable promoters (e.g., trp, lac, tac, X P L, etc.) and Shine-Dalgarno sequences will be fused with the entire coding sequence of those plasmids carrying an ATG codon preferably in front of the cleavage site of the signal peptide.
- suitable promoters e.g., trp, lac, tac, X P L, etc.
- Shine-Dalgarno sequences will be fused with the entire coding sequence of those plasmids carrying an ATG codon preferably in front of the cleavage site of the signal peptide.
- the cDNA clone of MCGF or multi-CSF, e.g., pcD-MCGF is first digested with PstI and X hoI endonuclease, and about 1 kb segment containing the entire protein coding sequence is subcloned into
- the segment can be subcloned into the PstI and SalI endonuclease sites of M13mp8.
- Single stranded M13mp8 DNA containing the complementary strand of protein coding sequence is annealed with a synthetic oligonucleotide (5' GAT ACC CAC CGT TTA 3'), and double-stranded protein coding sequence is then synthesized by the Klenow fragment.
- a blunt-ended DNA segment containing the double stranded MCGF coding sequence or multi-CSF coding sequence is inserted into an appropriate expression vector, such as pDR540, which has the tac promoter (see Russel, D.R and Bennett, G.N., Gene 20, 231-243 [1982]); and deBoer, H. et al., Proc. Natl. Acad. Sci. U.S.A. 80, 21-25 [1983]). (See generally Messing, J. et al., Proc. Nat. Acad. Sci. U.S.A. 74, 3642-3646 [1977]); Gronenborn, B.
- the PstI-XhoI fragment carrying a cDNA insert is isolated from pcD-MCGF plasmid, and then cloned into the PstI-SalI sites of pUC8.
- the resultant plasmid B8/pUC8 is cut with PstI and digested with Ba131 to remove the oligo (dG:dC) block placed upstream of the cDNA.
- An XhoI linker is attached to Bal31-digested DNA, and the plasmids are recovered in E. coli. The transformants are analysed to determine the size of the deletion.
- the XhoI-EcoRI fragment (carrying MCGF or multi-CSF cDNA) is then isolated from one of the deletion derivatives, which should have about a 20 base pair deletion, and cloned into the HindIII site of p AAH 5 and the EcoRI site of pAAR6 by blunt-end ligation using the Klenow fragment.
- pUC8 is an M13mp7-derived system useful for insertion mutagenesis and sequencing with synthetic universal primers: See Vieira, J. and Messing, J., Gene 19: 259-268 [1982]); for pcD-X, see Okayama, H. and Berg, P., Mol. Cell. Biol.
- pAAH5 and pAAR6 are yeast expression vectors carrying the ADCI promoter and terminator: Ammer, G., "Expression of Genes in Yeast using the ADCI promoter", Methods in Enzymology, 101: 192-201 [1982].)
- the multi-CSF and MCGF polypeptides expressed in E. coli, in yeast or in other cells can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fractionation column chromatography (e.g., ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and ultimately crystallization (see generally "Enzyme purification and Related Techniques", Methods in Enzymology, 22: 233-577 [1977]).
- the polypeptides of the invention may be used in pharmaceutical compositions (see below), e.g., for treating parasitic infections of the gastrointestinal tract; or for research purposes, e.g., as a supplement to hematopoietic or mast cell media and as an antigenic substance for eliciting specific immunoglobulins useful in immunoassays, immunofluorescent stainings, etc.
- pharmaceutical compositions see below
- immunoglobulins useful in immunoassays, immunofluorescent stainings, etc.
- compositions containing the polypeptides described by this invention are compounded by admixture with preferably inert, pharmaceutically acceptable carriers.
- Suitable carriers and processes for their preparation are well known in the art (see e.g. Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA [1980]).
- the preferred course of administration is parenteral and can include use of mechanical delivery systems.
- the pharmaceutical composition is in unit dosage form.
- the preparation is subdivided into unit doses containing appropriate quantities of the active component.
- the quantity of active compound in a unit dose of preparation may be varied or adjusted from 1 ug. to 100 mg., according to the particular application and the potency of the active ingredient.
- the composition can, if desired, also contain other therapeutic agents.
- the dosages may be varied depending upon the requirement of the patient, the severity of the condition being treated and the particular compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
- Frozen cell pellets from uninduced or ConA-induced Cl.Ly 1 + 2 - /9 were suspended in guanidine isothiocyanate lysis solution. Twenty ml of lysis solution was used for 1-2 x 10 8 cells. Pellets were resuspended by pipetting, then DNA was sheared by 4 passes through a syringe using a 16 gauge needle. The lysate was layered on top of 20 ml of 5.7 M CsCl, 10 mM EDTA in 40 ml polyallomer centrifuge tube.
- RNA pellets were washed twice with cold 70% ethanol. Pellets were then resuspended in 500 ⁇ l of 10 mM Tris ⁇ HCl pH 7.4, 1 mM EDTA, 0.05% SDS. 50 P l of 3M sodium acetate was added and RNA was precipitated with 1 ml ethanol. The RNA was collected by centrifuging and the pellets washed once with cold ethanol.
- RNA pellet was resuspended in 900 ⁇ l of oligo (dT) elution buffer (10 mM Tris ⁇ HCl, pH 7.4, 1 mM EDTA, 0.5% SDS). RNA was heated for 3 min. at 68°C and then chilled on ice. 100 ⁇ l of 5 M NaCl was added. The RNA sample was loaded onto a 1.0 ml oligo (dT) cellulose column (Type 3, Collaborative Research, Waltham, MA) equilibrated with binding buffer (10 mM Tris ⁇ HCl pH 7.4, 1 mM EDTA, 0.5 M NaCl, 0.5% SDS.). Flow-through from the column was passed over the column twice more.
- oligo (dT) elution buffer 10 mM Tris ⁇ HCl, pH 7.4, 1 mM EDTA, 0.5% SDS.
- RNA was precipitated with 0.1 volume 3 M sodium acetate (pH 6) and two volumes of ethanol. The RNA pellet was collected by centrifugation, washed twice with cold ethanol, and dried. The pellet was then resuspended in water. Aliquots were diluted, and absorbance at 260 nm was determined.
- the size-fractionated Poly A + mRNA following injection in Xenopus oocytes, gave a peak of MCGF activity by the colorimetric assay sedimenting slower than 18S, as shown in Figure 2. These fractions were enriched approximately 10-fold for MCGF mRNA and were utilized subsequently for the preparation of 32p- labelled cDNA probe.
- Oocytes were removed from female Xenopus laevis and incubated in Barth's solution (88 mM NaCl, 1 mM KC1, 0.33 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , 2.4 mM NaHC0 3 , 10 mM HEPES (pH 7.9) (Sigma Chemical Co., St. Louis, MO). Injection clusters of 2-3 oocytes were prepared. RNA samples were to be injected dissolved in injection buffer (40 mM Tris ⁇ HCl pH 7.4, 0.35 M NaCl).
- RNA samples eluted from DNA filters from hybrid selections always contained 5 Pg of calf liver tRNA as carrier and were resuspended in 2 ⁇ l of injection buffer.
- 40 nl aliquots were injected into each oocyte using micropipets pulled by hand with tips forged using a microforge. The pipettes were calibrated with known volumes of sterile water. Approximately 30-40 oocytes were injected for each mRNA sample.
- the injected oocytes were incubated in groups of two or three in individual wells of 96-well microtiter dishes containing 10 P l of Barth's solution + 1% bovine serum albumin per oocyte.
- the oocytes were kept at 19°C for 48 hours and then the supernatants from wells containing viable oocytes were collected and pooled. These supernatants were sterilized by centrifuging for 10 minutes in a microcentrifuge and then assayed for MCGF activity as described above. Supernatants from uninjected oocytes were always collected as a control.
- phenol-CHC1 3 water-saturated 1:1 phenol-CHC1 3 (hereafter referred to as phenol-CHC1 3 ) and ethanol precipitation.
- the reaction mixture (38 ⁇ l) contained sodium cacodylate-30 mM Tris ⁇ HCl pH 6.8 as buffer, with 1 mM CoCl 2 , 0.1 mM dithiothreitol, 0.25 mM dTTP, the KpnI endonuclease-digested DNA, and 68 U of the terminal deoxynucleotidyl transferase (P-L Biochemicals, Inc., Milwaukee, WI).
- dT deoxythymidylate
- the reaction was stopped with 20 ⁇ l of 0.25 M EDTA (pH 8.0) and 10 ⁇ l of 10% SDS, and the DNA was recovered after several extractions with phenol-CHC1 3 by ethanol precipitation.
- the DNA was then digested with 15 U of EcoRI endonuclease in 50 ⁇ l containing 10 mM Tris ⁇ HCl pH 7.4, 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.1 mg of BSA per ml for 5 hr at 37°C.
- the large fragment containing the SV40 polyadenylation site and the pBR322 origin of replication and ampicillin- resistance gene, was purified by agarose (1%) gel electrophoresis and recovered from the gel by a modification of the glass powder method (Vogelstein, B. & Gillespie, D., Proc. Nat. Acad. Sci. 76: 615-619 [1979]).
- the dT-tailed DNA was further purified by absorption and elution from an oligo (dA)-cellulose column as follows: The DNA was dissolved in 1 ml of 10 mM Tris ⁇ HCl pH 7.3 buffer containing 1 mM EDTA and 1 M NaCl, cooled at 0°C, and applied to an oligo (dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same buffer at 0°C. The column was washed with the same buffer at 0°C and eluted with water at room temperature. The eluted DNA was precipitated with ethanol and dissolved in 10 mM Tris ⁇ HCl pH 7.3 with 1 mM EDTA.
- the oligo (dG) tailed linker DNA was prepared by digesting 75 Pg of pL1 DNA with 20 U of PstI endonuclease in 450 ⁇ l containing 6 mM Tris ⁇ HCl pH 7.4, 6 mM MgCl 2 , 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per ml. After 16 hr at 30°C the reaction mixture was extracted with phenol-CHC1 3 and the DNA was precipitated with alcohol.
- Tails of 10 to 15 deoxyguanylate (dG) residues were then added per end with 46 U of terminal deoxynucleotidyl transferase in the same reaction mixture (38 Pl) as described above, except that 0.1 mM dGTP replaced dTTP.
- the mixture was extracted with phenol-CHCl 3 , and after the DNA was precipitated with ethanol it was digested with 35 U of HindIII endonuclease in 50 ⁇ l containing 20 mM Tris ⁇ HCl pH 7.4, 7 mM MgCl 2 , 60 mM NaCl, and 0.1 mg of BS A at 37°C for 4 hr.
- the small oligo (dG)-tailed linker DNA was purified by agarose gel (1.8%) electrophoresis and recovered as described above.
- Step 1 cDNA synthesis.
- the reaction mixture (10 ⁇ l) contained 50 mM Tris ⁇ HCl pH 8.3, 8 mM MgCl 2 , 30 mM KCl, 0.3 mM dithiothreitol, 2 mM each dATP, dTTP, dGTP, and dCTP, 20 ⁇ Ci 32 P -dCTP (3000 Ci/mmole), 2 ⁇ g polyA + RNA from Con-A induced Cl.Ly 1 + 2 - /9, 60 units RNase (Biotec, Inc., Madison, WI), and 2 ⁇ g of the vector-primer DNA (15 pmol of primer end), and 45 U of reverse transcriptase.
- the reaction was incubated 60 min at 42°C and then stopped by the addition of 1 Pl of 0.25 M ETDA (pH 8.0) and 0.5 ⁇ l of 10% SDS; 40 ⁇ l of phenol-CHC1 3 was added, and the solution was blended vigorously in a Vortex mixer and then centrifuged. After adding 40 ⁇ l of 4 M ammonium acetate and 160 ⁇ l of ethanol to the aqueous phase, the solution was chilled with dry ice for 15 min., warmed to room temperature with gentle shaking to dissolve unreacted deoxynucleoside triphosphates that had precipitated during chilling, and centrifuged for 10 min. in an Eppendorf microfuge.
- the pellet was dissolved in 10 ⁇ l of 10mM Tris ⁇ HCl pH 7.3 and 1 mM EDTA, mixed with 10 ⁇ l of 4 M ammonium acetate, and reprecipitated with 40 ⁇ l of ethanol, a procedure which removes more than 99% of unreacted deoxynucleoside triphosphates.
- the pellet was rinsed with ethanol.
- Step 2 Oligodeoxycytidylate [oligo (dC)] addition.
- the pellet containing the plasmid-cDNA:mRNA was dissolved in 20 ⁇ l of 140 mM sodium cacodylate-30 mM Tris ⁇ HCl pH 6.8 buffer containing 1 mM CoCl 2 , 0.1 mM dithiothreitol, 0.2 P g of poly(A), 70 ⁇ M dCTP, 5 ⁇ Ci 32 P-dCTP, 3000 Ci/mmole, and 60 U of terminal deoxynucleotidyl transferase. The reaction was carried out at 37°C for 5 min.
- Step 3 HindIII endonuclease digestion.
- the pellet was dissolved in 30 ⁇ l of buffer containing 20 mM Tris ⁇ HCl pH 7.4, 7 mM MgCl 2 , 60 mM NaCl, and 0.1 mg of BSA per ml and then digested with 10 U of HindIII endonuclease for 2 hr at 37°C.
- the reaction was terminated with 3 Pl of 0.25 M EDTA (pH 8.0) and 1.5 ⁇ l of 10% SDS and, after extraction with phenol-CHC1 3 followed by the addition of 30 ⁇ l of 4 M ammonium acetate, the DNA was precipitated with 120 ⁇ l of ethanol.
- the pellet was rinsed with ethanol and then dissolved in 10 Pl of 10 mM Tris ⁇ HCl (pH 7.3) and 1 mM EDTA, and 3 ⁇ l of ethanol was added to prevent freezing during storage at -20°C.
- Step 4 Cyclization mediated by the oligo (dG)-tailed linker DNA.
- the mixture (90 ⁇ l) was adjusted to a volume of 900 ⁇ l containing 20 mM Tris ⁇ HCl pH 7.5, 4 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 0.1 M KC1, 50 ⁇ g of BSA per ml, and 0.1 mM S-NAD; 6Pg of E. coli DNA ligase were added and the solution was then incubated overnight at 12°C.
- Step 5 Replacement of RNA strand by DNA.
- the ligation mixture was adjusted to contain 40 ⁇ M of each of the four deoxynucleoside triphosphates, 0.15 mM S-NAD, 4 ⁇ g of additional E. coli DNA ligase, 16 U of E. coli DNA polymerase I (PolI,) and 9 U of E. coli RNase H. This mixture (960 ⁇ l) was incubated successively at 12°C and room temperature for 1 hr each to promote optimal repair synthesis and nick translation by Poll.
- Step 6 Transformation of E. coli.
- E. coli K-12 strain MC1061 (Casadaban, M. and Cohen, S., J. Mol. Biol. 138: 179-207 [1980]) was grown to 0.5 absorbancy unit at 600 nm at 37°C in 20 ml of L-broth. The cells were collected by centrifugation, suspended in 10 ml of 10 mM Tris ⁇ HCl pH 7.3 containing 50 mM CaCl 2 , and centrifuged at 0°C for 5 min.
- the cells were resuspended in 2 ml of the above buffer and incubated again at 0°C for 5 min.; then, 0.2 ml of the cell suspensions was mixed with 0.1 ml of the DNA solution (step 5) and incubated at 0°C for 15 min. Next the cells were kept at 37°C for 2 min. and thereafter at room temperature for 10 min.; then 0.5 ml of L-broth was added, and the culture was incubated at 37°C for 30 min., mixed with 2.5 ml of L-broth soft agar at 42°C, and spread over L-broth agar containing 50 Pg of ampicillin per ml. After incubation at 37°C for 12 to 24 hr, individual colonies were picked with sterile tooth-picks.
- Hybrid selections were performed with eight cDNA plasmid preparations, taken from the random pools described above.
- All plasmid DNAs were linearized by digestion with ClaI prior to binding to nitrocellulose filters. Digestions were performed in 50 ⁇ l containing: 10 mM Tris ⁇ HCl pH 7.9, 10 mM MgCl 2 , 10 Pg plasmid DNA, 50 mM NaCl, and 10 units ClaI. Following a 2 hr incubation at 37°C, samples were diluted to 200 ⁇ l with TE (10 mM Tris ⁇ HCl pH 8.0, 1 mM EDTA) and extracted with an equal volume (200 ⁇ l) of phenol saturated with TE. 20 Pl of 3M sodium acetate (pH 6) was added to the aqueous phase, and this was precipitated with 2 volumes of ethanol.
- the DN A pellets were recovered by centrifugation and then washed with 70% ethanol. The dried pellet was resuspended in 150 ⁇ l of sterile water for each 10 ⁇ l of DNA. Duplicate filters were prepared for each DNA sample, 10 ⁇ g DNA per filter. The DNA in 150 ul of water was boiled for 10 min, then 150 ⁇ l 1N NaOH was added and the solution incubated 20 min at room temperature. The sample was chilled on ice, then 150 ⁇ l 1M HC1, 1M NaCl, 0.3M Na-citrate and 0.5M Tris ⁇ HCl pH 8.0 was added.
- Millipore HAWP filters wet with distilled water were fitted into Schleicher-and-Schuell microfiltration apparatus. The denatured and neutralized DNA solution from above was filtered through by centrifugation at 500 rpm for 5 min. Filters were washed with 1 ml of 6xSSC (0.15 M NaCl, 0.015 M Na citrate) and then air-dried before baking 2 hrs. at 80°C.
- Hybridizations were performed in 200 ⁇ l containing 65% (v/v) redistilled formamide, 20 mM PIPES, pH 6.4, 0.4 M NaCl, 200 ⁇ g/ml calf liver tRNA, and 100 Pg/ml polyA + mRNA from ConA-induced Cl.Ly 1 + 2 - /9. Each hybridization solution was heated for 3 min at 70°C and then two DNA filters (10 Pg DNA/filter) were cut into quarters and added to the solution. Hybrids were incubated at 50°C for 4 hours followed by 4 hour incubations at 46° and 42°C.
- RNA pellets were collected by centrifugation and washed twice with 70% ethanol. The dried pellets were resuspended in 2 ⁇ l of oocyte injection buffer and the entire sample was injected into oocytes (see above).
- 32 P-cDNA (synthesized as described above) was co-precipitated with 20 ⁇ g of polyA + mRNA from WEHI-3 and 20 ⁇ l of poly A+ mRNA from a B-cell hybridoma.
- the hybridization mixture was then diluted to 1 ml with 0.12 M sodium phosphate pH 7.0 and 0.1% SDS, and the temperature of the mixture raised to 60°C.
- the mixture was then loaded on a column of 0.4 gm hydroxyapatite equilibrated in the same buffer and kept at 60°C. The flowthrough was collected and the column was then washed with 5 ml of the same buffer at 60°C. 1 ml fractions were collected and 1 ⁇ l aliquots of each fraction were counted in a scintillation counter. The peak of single stranded cDNA in fractions 2, 3, and 4 was pooled. This material, representing 66.5% of the starting 32 P-cDNA, was concentrated to 0.4 ml by extraction with 2-butanol and then desalted by chromatography on a 2 ml Sephadex G-25 column.
- the desalted sample from above was concentrated by ethanol precipitation and then co-precipitated with 9.5 ⁇ g of poly A+ mRNA from uninduced Cl.Ly 1 + 2 - /9.
- the washed and dried pellet was resuspended in 10 ul water, 1.5 ⁇ l 4 M sodium phosphate pH 7, 0.15 ⁇ l 20% SDS and 0.15 ⁇ l 0.1 M EDTA.
- the sample was incubated in a sealed capillary tube for 30 min at 90°C and then at 68°C for 20 hr. Chromatography on hydroxyapatite was repeated as described above.
- the single stranded cDNA which eluted from the column at 60°C represented 17% of the starting material.
- This 32 P-cDNA was used for colony hybridizations of the 48 colonies in the sub-pool identified by hybrid selections. Three colonies hybridized with the probe and were used for further hybrid selection. One of these, designated clone 5G, was reproducibly positive.
- plasmid DNA representing the entire cDNA library (pcD-X DNA) was digested separately with the restriction enxymes SalI, HindIII, and ClaI to linearize the plasmid.
- the restricted DNAs were size-fractionated on a 1% agarose gel to separate plasmids having different size cDNA inserts. Segments were excised from the gel representing plasmids with cDNA inserts of the following size ranges:
- DNA was eluted from each gel slice using the glass powder method of Vogelstein and Gillespie (Proc. Nat. Acad. Sci. U.S.A., 76: 615-619 [1970]).
- the eluted DNAs from the 3 digests were pooled on the basis of size, and treated with T4 ligase to recyclize in a total volume of 15 ul containing 50 mM Tris ⁇ HCl pH 7.4, 10 mM MgCl 2 , 10 mM DTT, 1 mM spermidine, 1 mM ATP and 100 Pg/ml BSA.
- the ligation reactions were incubated 16 hr at 12°C. 3 ul of each combined size fraction was used to transform E.
- a plasmid (pcD-MCGF) carrying a full-length MCGF and multi-CSF (see below) cDNA insert is shown in Figure 3, and an E. coli bacterium carrying the plasmid has been deposited with the ATCC (accession number 39467).
- the 950 bp insert is contained in the pcD expression vector. Transcription from the SV40 early promoter is indicated by the arrow. The location of the splice donor and acceptor sites are shown.
- the polyadenylation signal, also derived from SV40, is located at the 3' end of the cDNA insert. The cDNA insert is heavily shaded.
- Figure 4 shows the restriction endonuclease cleavage map of the cDNA insert of the present invention, and Figure 1 contains the nucleotide sequence and putative amino acid sequence.
- Three cDNA inserts contain a single open reading frame consisting of 166 codons beginning with the methionine codon at position 28. In addition to this putative initiation codon, two other methionine codons occur, 12 and 18 codons downstream from the first.
- a fourth cDNA clone starts 40 base pairs downstream from the 5' ends of the other three inserts. This shorter cDNA clone lacks the first methionine codon but still makes active MCGF upon introduction into COS cells. Thus, one of the two ATG codons downstream can apparently serve as the initiation codon.
- COS-MCGF COS-7 cells
- the expressed B-9 clone was tested under conditions which allow generation of BFU-E, CFU-G/M and CFU-Mixed.
- Table III shows that three types of colonies could be identified and enumerated in cultures of bone marrow cells incubated with the expressed material. The most prevalent type consisted of colorless colonies lacking hemoglobinized elements. Their morphology was typical of granulocyte/macrophage colonies, the existence of which was later confirmed by histochemical staining. Also present were some large macroscopic colonies containing a multicentric arrangement of uniformly red cell clusters, which were designated BFU-E. We further observed a few colonies containing hemoglobinized cells mixed with large and small translucent cells, which were counted as mixed.
- composition of these various colonies was analyzed by applying selected colonies to glass slides and staining with Wright-Giemsa or nonspecific esterase stains. Over 300 colonies were examined. The majority of these colonies (89%) were composed of granulocytes, macrophages or a granulocyte/macrophage mixture, while four percent consisted of mast cells. The remainder were composed of mixed lineages other than neutrophil/macrophage. Differential counts of 10 representative mixed colonies compiled from several experiments are presented in Table IV. The presence of several cell types within single colonies suggests that these colonies derive from pluripotent progenitor cells.
- COS-MCGF has erythroid burst-promoting activity and allows expansion of stem cells and early committed progenitor cells of several lineages, including monocytic/granulocytic, erythroid and megakaryocytic cells. This range of activities indicates that the cDNA clones of the present invention encode proteins having the characteristics of growth factors for hematopoietic cells for multiple lineages.
- the cDNA clones of the present invention provide accurate and complete sequence data on mammalian multi-CSF and mast cell growth factors.
- the invention also provides to those skilled in the art means for producing significant quantities of such factors (essentially free from other hematopoietic factors) for the improved in vitro maintenance of mast cells and other hematopoietic cells. Further, the information gleaned from the cDNA clones increases understanding of the mammalian immune response, enhancing therapeutic potentialities.
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AT84111677T ATE74963T1 (de) | 1983-10-04 | 1984-09-29 | Cdna-klone, codierend fuer polypeptide mit der wirksamkeit eines nicht-zellinienspezifischen wachstumsfaktors (multi-csf) und/oder mastzellenwachstumsfaktors (mcgf). |
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US53905083A | 1983-10-04 | 1983-10-04 | |
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US06/590,867 US4695542A (en) | 1983-10-04 | 1984-03-19 | cDNA clones coding for polypeptides exhibiting multi-lineage cellular growth factor activity |
US590867 | 1984-03-19 |
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US6238889B1 (en) | 1986-12-16 | 2001-05-29 | Dsm N.V. | Molecular cloning and expression of the Pro8 isoform of human IL-3 |
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NZ210501A (en) * | 1983-12-13 | 1991-08-27 | Kirin Amgen Inc | Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence |
US5955315A (en) * | 1985-11-19 | 1999-09-21 | Schering Corporation | Nucleic acids encoding human interleukin-4 |
US4959455A (en) * | 1986-07-14 | 1990-09-25 | Genetics Institute, Inc. | Primate hematopoietic growth factors IL-3 and pharmaceutical compositions |
GR871029B (en) * | 1986-07-14 | 1987-11-02 | Genetics Inst | Novel osteoinductive factors |
US5047335A (en) * | 1988-12-21 | 1991-09-10 | The Regents Of The University Of Calif. | Process for controlling intracellular glycosylation of proteins |
EP0466780A4 (en) * | 1989-04-03 | 1992-08-26 | Genetics Institute, Inc. | Megakaryocyte growth promoting activity |
US5260417A (en) * | 1989-04-03 | 1993-11-09 | Genetics Institute, Inc. | Megakaryocyte growth promoting activity protein |
US20040181044A1 (en) * | 1989-10-16 | 2004-09-16 | Zsebo Krisztina M. | Method of stimulating growth of epithelial cells by administering stem cell factor |
HU220234B (hu) * | 1989-10-16 | 2001-11-28 | Amgen Inc. | Eljárás stem sejtek működését befolyásoló faktorok előállitására |
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US7144731B2 (en) * | 1989-10-16 | 2006-12-05 | Amgen Inc. | SCF antibody compositions and methods of using the same |
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- 1984-09-29 EP EP84111677A patent/EP0138133B1/fr not_active Expired - Lifetime
- 1984-09-29 DE DE8484111677T patent/DE3485652D1/de not_active Expired - Fee Related
- 1984-10-01 DK DK470184A patent/DK470184A/da not_active Application Discontinuation
- 1984-10-01 NZ NZ209734A patent/NZ209734A/en unknown
- 1984-10-01 FI FI843844A patent/FI87802C/fi not_active IP Right Cessation
- 1984-10-01 GR GR80517A patent/GR80517B/el unknown
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- 1984-10-02 GB GB8424832A patent/GB2147585B/en not_active Expired - Fee Related
- 1984-10-02 KR KR1019840006097A patent/KR850003164A/ko not_active Application Discontinuation
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EP0167548A4 (fr) * | 1983-12-23 | 1988-04-26 | Univ Australian | CLONAGE DE cADN ET SECRETION DE MURINE-INTERLEUCINE-3. |
WO1987002990A1 (fr) * | 1985-11-19 | 1987-05-21 | Schering-Biotech Corporation | Interleukine-4 mammifere |
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EP0675136A3 (fr) * | 1985-11-19 | 1996-03-13 | Schering Biotech Corp | Anticorps contre l'interleukine-4 de mammifères et peptides utiles comme antigènes pour leur préparation. |
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US6238889B1 (en) | 1986-12-16 | 2001-05-29 | Dsm N.V. | Molecular cloning and expression of the Pro8 isoform of human IL-3 |
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US5304637A (en) * | 1987-07-13 | 1994-04-19 | Gist-Brocades N.V. | Expression and purification of human interleukin-3 and muteins thereof |
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US5128450A (en) * | 1989-06-30 | 1992-07-07 | Urdal David L | Nonglycosylated human interleukin-3 analog proteins |
EP0909817A1 (fr) * | 1990-09-28 | 1999-04-21 | Chiron Corporation | Utilisations des gènes codant pour GRO et des protéines |
US5994060A (en) * | 1990-09-28 | 1999-11-30 | Chiron Corporation | Gro genes, proteins, and uses thereof |
WO1992006196A1 (fr) * | 1990-09-28 | 1992-04-16 | Cetus Oncology Corporation | Genes et proteines gro, ainsi que leur utilisation |
US7220828B2 (en) * | 1995-10-23 | 2007-05-22 | Zenyth Operations Pty Ltd | Haemopoietin receptor and genetic sequence encoding same |
Also Published As
Publication number | Publication date |
---|---|
ATE74963T1 (de) | 1992-05-15 |
FI843844A0 (fi) | 1984-10-01 |
HU204088B (en) | 1991-11-28 |
IL73111A (en) | 1992-11-15 |
DE3485652D1 (de) | 1992-05-21 |
DK470184A (da) | 1985-04-05 |
FI843844L (fi) | 1985-04-05 |
GB2147585B (en) | 1990-12-12 |
HUT36860A (en) | 1985-10-28 |
DK470184D0 (da) | 1984-10-01 |
EP0138133B1 (fr) | 1992-04-15 |
GB2147585A (en) | 1985-05-15 |
FI87802B (fi) | 1992-11-13 |
US4695542A (en) | 1987-09-22 |
AU3375684A (en) | 1985-04-18 |
GB8424832D0 (en) | 1984-11-07 |
NZ209734A (en) | 1992-08-26 |
GR80517B (en) | 1985-02-05 |
KR850003164A (ko) | 1985-06-13 |
FI87802C (fi) | 1993-02-25 |
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